Mechanisms of Flavoproteins

描述(申请人提供)：黄蛋白在整个新陈代谢过程中被发现，催化细胞中的各种关键步骤。在这些酶进行的反应中，有许多是未活化的碳氢键断裂的。这是生物系统中的一种基本反应，由多种酶类催化。本提案的总体目标是了解黄素蛋白催化碳氢键断裂反应的机制，并确定不同蛋白质折叠利用相同机制催化这些反应的程度。这些实验的重点是氧化碳和氧或氮之间的键的酶，转移相当于黄素辅因子的氢化物。选择了代表不同底物类别的酶进行研究：D-氨基酸氧化酶和色氨酸单加氧酶作为D-氨基酸氧化酶和L-氨基酸氧化酶，黄色素b2作为羟基酸氧化酶，以及硝基烷氧化酶，这是研究黄素蛋白形成碳负离子的独特机会。为了阐明每个酶的碳氢键断裂步骤的细节，人们提出了各种机械实验。这些都与突变分析相结合，旨在确定蛋白质折叠与机制相关的程度。这些研究的结果将为深入了解其他黄素蛋白的机制，蛋白质在引导黄素辅助因子的反应中的作用，以及生物系统裂解碳-氢键的一般问题提供帮助。

DESCRIPTION (provided by applicant): Flavoproteins are found throughout metabolism, catalyzing a variety of critical steps in the cell. Among the reactions which these enzymes carry out are many in which an unactivated carbon-hydrogen bond is cleaved. This is a fundamental reaction in biological systems, catalyzed by a variety of enzyme classes. The overall goal of the present proposal is to understand the mechanisms by which flavoproteins catalyze carbon-hydrogen bond cleavage reactions and to determine the extent to which different protein folds utilize the same mechanism to catalyze these reactions. The experiments focus on enzymes which oxidize bonds between carbon and either oxygen or nitrogen, transferring a hydride equivalent to the flavin cofactor. Enzymes representative of different classes of substrates have been selected for study: D-amino acid oxidase and tryptophan monooxygenase as D- and L-amino acid oxidases, flavocytochrome b2 as a hydroxy acid oxidizing enzyme, and nitroalkane oxidase, a novel enzyme which represents a unique opportunity to study carbanion formation by a flavoprotein. A variety of mechanistic experiments are proposed to elucidate the details of the carbon-hydrogen bond cleavage step by each enzyme. These are combined with mutational analyses designed to determine the extent to which the protein fold correlates with mechanism. The results of these studies will provide insight into the mechanisms of other flavoproteins, into the roles of the protein in directing the reactivity of the flavin cofactor and into the general problem of cleavage of carbon-hydrogen bonds by biological systems.